The Cathepsin B of Toxoplasma gondii,Toxopain-1, Is Critical for Parasite Invasion and Rhoptry Protein Processing

Abstract

Cysteine proteinases play a major role in invasion and intracellular survival of a number of pathogenic parasites. We cloned a single copy gene, tgcp1, from Toxoplasma gondii and refolded recombinant enzyme to yield active proteinase. Substrate specificity of the enzyme and homology modeling identified the proteinase as a cathepsin B. Specific cysteine proteinase inhibitors interrupted invasion by tachyzoites. The T. gondii cathepsin B localized to rhoptries, secretory organelles required for parasite invasion into cells. Processing of the pro-rhoptry protein 2 to mature rhoptry proteins was delayed by incubation of extracellular parasites with a cathepsin B inhibitor prior to pulse-chase immunoprecipitation. Delivery of cathepsin B to mature rhoptries was impaired in organisms with disruptions in rhoptry formation by expression of a dominant negative 1-adaptin. Similar disruption of rhoptry formation was observed when infected fibroblasts were treated with a specific inhibitor of cathepsin B, generating small and poorly developed rhoptries. This first evidence for localization of a cysteine proteinase to the unusual rhoptry secretory organelle of an apicomplexan parasite suggests that the rhoptries may be a prototype of a lysosome-related organelle and provides a critical link between cysteine proteinases and parasite invasion for this class of organism.The protozoan, Toxoplasma gondii, is an obligate intracellular parasite that can invade and replicate within any nucleated cell of vertebrate hosts, including humans (1-3). Invasion by T. gondii tachyzoites is mediated by the sequential regulated release of specialized secretory organelles of the parasite including the micronemes, rhoptries, and dense granules (4). In early observations, the penetration of host cells by tachyzoites was enhanced by the addition of partially purified lysosomal enzymes (5). In addition, proteinases have been implicated in host cell invasion in other members of the Apicomplexa such as Plasmodium (6) and Eimeria tenella (7). We now show that a cathepsin B, toxopain-1, is strongly implicated in T. gondii invasion and that infection can be interrupted with specific cathepsin B inhibitors.Another unusual feature of Toxoplasma is the absence of a morphologically identifiable lysosomal system. In higher eukaryotic cells, acidic cathepsins in lysosomes are important in protein processing and breakdown. The mammalian precursor of cathepsin B is targeted to the lysosomal compartment by mannose 6-P for proteolytic activation (8). Thus, the apparent lack of a lysosomal system in Toxoplasma raises a number of questions regarding cellular proteinase functions within the parasite. The rhoptries are club-shaped organelles located at the apical end of the parasites with no known counterpart outside of the phylum Apicomplexa (9). Although nine rhoptry proteins (ROP1-9) 1 have been identified to date (10), a definitive function has only been determined for ROP2, which mediates binding of host mitochondria to the parasitophorous membrane (11). How rho...

Cathepsin Cs Are Key for the Intracellular Survival of the Protozoan Parasite, Toxoplasma gondii

“…The remaining sequences were obtained by 3Ј-and 5Ј-rapid amplification of cDNA ends and cloned into the TA vector and sequenced (12). All nucleotide sequences and predicted amino acid sequences were compared using the BLAST programs at the National Center for Biotechnology Information.…”

“…Proteins were expressed in E. coli as C-terminally His 6 -tagged recombinant protein and purified on a nickel-nitrilotriacetic acid-agarose resin column as described previously (12).…”

“…Assays of Cysteine Proteinase Activity-Proteinase activity was assayed by the liberation of the fluorescent leaving group, AMC, and measured as the increase in fluorescence using an automated microtiter plate spectrofluorometer (LabSystems Fluoroskan II) as described previously (12). The initial velocities were calculated by linear regression of the substrate hydrolysis curves.…”

“…Human cathepsin C plays a role in lysosomal degradation of peptides and also functions as a key enzyme in the activation of granule serine proteases in human and murine immune cells (7)(8)(9)(10)(11). We recently char-acterized the T. gondii cathepsin B (toxopain-1 or TgCPB) 2 (12) and cathepsin L (TgCPL) proteases. TgCPB localizes to the rhoptry organelles, which are critical to host invasion (12).…”

“…We recently char-acterized the T. gondii cathepsin B (toxopain-1 or TgCPB) 2 (12) and cathepsin L (TgCPL) proteases. TgCPB localizes to the rhoptry organelles, which are critical to host invasion (12). Based on specificity differences from host enzymes and their critical role in peptide degradation, Toxoplasma cathepsin Cs play important roles in tachyzoite growth and differentiation and may be exploited as drug targets in the future.…”